This invention relates to compounds and methods for inducing or promoting apoptosis and for arresting uncontrolled neoplastic cell proliferation, methods that are specifically useful in the arresting and treatment of neoplasias, including precancerous and cancerous lesions.
Pharmaceuticals that are effective against early stage neoplasias comprise an emerging and expanding area of research and potential commercial development. Such pharmaceuticals can delay or arrest development of precancerous lesions into cancers. Each year in the United States alone, untold numbers of people develop precancerous lesions, which exhibit a strong statistically significant tendency to develop into malignant tumors, or cancer. Such lesions include lesions of the breast (that can develop into breast cancer), lesions of the skin (that can develop into malignant melanoma or basal cell carcinoma), colonic adenomatous polyps (that can develop into colon cancer), cervical dysplasia (cervical cancer) and other such neoplasms.
Such compounds and methods are particularly beneficial to sub-populations of patients who repeatedly develop precancerous lesions, and therefore have a statistically higher probability of getting cancer. Many cancer types (e.g., breast, colon, prostate etc.) have such patient sub-populations.
The search for drugs useful for treating and preventing neoplasias in their earliest stages is intensive because chemotherapy and surgery on cancer itself is often not effective, and current cancer chemotherapy has severe side effects. Such cancer-preventative compounds are also envisaged for recovered cancer patients who retain a risk of cancer reoccurrence, and even for cancer patients who would benefit from compounds that selectively induce apoptosis in neoplastic, but substantially not in normal cells.
Because it is believed that chronic administration of cancer-preventative pharmaceuticals is necessary to inhibit or arrest the development of neoplasia, standard cancer chemotherapeutic drugs are not considered appropriate drugs for cancer chemoprevention because whatever cancer preventative (as opposed to cancer-fighting) capabilities those drugs may possess do not outweigh their severe side effects. Most standard chemotherapeutics are now believed to kill cancer cells by inducing apoptosis (also sometimes referred to as xe2x80x9cprogrammed cell deathxe2x80x9d). Apoptosis naturally occurs in many tissues in the body. Apoptosis plays a critical role in tissue homeostasis, that is, it ensures that the number of new cells produced are correspondingly offset by an equal number of cells that die. Apoptosis is especially pronounced in self-renewing tissues such as bone marrow, immune cells, gut, and skin. For example, the cells in the intestinal lining divide so rapidly that the body must eliminate cells after only three days to protect and prevent the overgrowth of the intestinal lining.
Standard chemotherapeutics promote apoptosis not only in cancer cells, but also in normal human tissues, and therefore have a particularly severe effect on tissues where apoptosis is especially pronounced (e.g. hair, gut and skin). The results of those effects include hair loss, weight loss, vomiting and bone marrow immune suppression. Thus, standard chemotherapeutics are inappropriate for cancer prevention, particularly if chronic administration is indicated.
Several non-steroidal anti-inflammatory drugs (xe2x80x9cNSAIDsxe2x80x9d), originally developed to treat arthritis, have shown effectiveness in inhibiting and eliminating colonic polyps. Polyps virtually disappear when the patients take the drug, particularly when the NSAID sulindac is administered. However, the continued prophylactic use of currently available NSAIDs, even in high colon cancer-risk patients, is still marked by severe side reactions that include gastrointestinal irritations, perforations, ulcerations and kidney toxicity believed to be produced by inhibition of prostaglandin synthetase activity (xe2x80x9cPGE-2xe2x80x9d). Such inhibition is a requirement for the NSAIDs anti-inflammatory action since elevated levels of PGE-2 are associated with inflammation. PGE-2 plays a protective function in the gastrointestinal tract, which is the reason such gastric side effects arise with chronic NSAID therapy, which is rarely indicated for arthritis sufferers, acute therapy being the norm for them. However, chronic administration of sulindac is important for high cancer-risk patients to eliminate and prevent future polyps which cause gastric side effects in many such patients. Once NSAID treatment is terminated due to such complications, the neoplasms return, particularly in high risk patients.
Compounds such as those disclosed in U.S. Pat. No. 5,643,959 have exhibited advantages in the treatment of neoplastic lesions since such compounds have been shown to induce apoptosis in neoplastic cells but not in normal cells in humans (see Piazza et al. Gastroenterology Vol. 112, A629, 1997). Thus, the severe side effects due to induction of apoptosis in normal cells by conventional chemotherapeutics are avoided by these novel therapeutics (see, Piazza et al. Cancer Research Vol. 57, pp. 2452-2459, 1997). In addition, such compounds do not exhibit the gastric side effects associated with NSAIDs since such compounds do not substantially inhibit PGE-2. More potent compounds with such neoplasia specificity but without substantial PGE-2 activity are desirable.
This invention represents potent compounds that inhibit the growth of neoplastic cells, for treating patients with neoplastic lesions. This invention also involves methods for inducing such specific inhibition of neoplastic cells by exposing such cells to a pharmacologically effective amount of those compounds described below to a patient in need of such treatment. Such compositions are effective in modulating the growth of neoplasms.
As discussed above, the present invention includes compounds of Formula I below (as well as their pharmaceutically acceptable salts) for treating a patient with neoplastic, particularly precancerous, and cancerous lesions: 
wherein R1 and R2 are independently selected from the group consisting of hydrogen, lower alkyl, and benzyl;
R3 is selected from the group consisting of substituted or unsubstituted phenyl, benzyl, pyridinyl, pyrrolyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolidinyl, piperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, tetrazolyl, morpholinyl, triazinyl, furfuryl, and thiophenyl, and lower alkyl, wherein said substituents are one to three independently selected from the group consisting of halogen, lower alkyl, lower alkoxy, amino, lower alkylamino, di-lower alkylamino, hydroxy, nitro, nitrile, carboxyl, aminosulfonyl, lower alkyl mercapto, and lower alkylsulfonyl;
R4 is selected from the group consisting of substituted or unsubstituted phenyl, benzyl, pyridinyl, pyrrolyl, pyrrolidinyl, pyrazolyl, pyrazolidinyl, imidazolyl, imidazolidinyl, piperidinyl, pyrazinyl, piperazinyl, pyrimidinyl, tetrazolyl, morpholinyl, triazinyl, furfuryl, thiophenyl, and lower alkyl; wherein said substituents are one to three independently selected from the group consisting of halogen, lower alkyl, lower alkoxy, amino, lower alkylamino, di-lower alkylamino, hydroxy, nitro, nitrile, carboxyl, aminosulfonyl, lower alkyl mercapto, and lower alkylsulfonyl;
R5 is selected from the group consisting of hydrogen, lower alkyl, halogen, hydroxy, amino, lower alkyl amino, and dilower alkylamino;
Y is selected from the group consisting of CH2, Cxe2x95x90O, CHxe2x80x94OH
m is an integer from 0-3
X is selected from the group consisting of CH2, Cxe2x95x90O, CHxe2x80x94OH, and SO2; and
n is an integer from 0-2.
Preferred compounds of this invention include those where
R1 and R2 are independently selected from the group consisting of hydrogen and lower alkyl;
R3 is selected from the group consisting of substituted or unsubstituted phenyl, pyridinyl, pyrrolyl, pyrazolyl, imidazolyl, pyrazinyl, pyrimidinyl, triazinyl, furfuryl, thiophenyl, and lower alkyl, wherein said substituents are one to three independently selected from the group consisting of lower alkyl, lower alkoxy, amino, lower alkylamino, and di-lower alkylamino, carboxyl, aminosulfonyl and alkylsulfonyl.
R4 is selected from the group consisting of substituted or unsubstituted phenyl, pyridinyl, pyrroyl, imidazolidinyl, pyrazinyl, piperazinyl, pyrimidinyl, morpholinyl, triazinyl, thiophenyl, and lower alkyl, wherein said substituents are one to three independently selected from the group consisting of lower alkoxy, amino, di-lower-alkylamino, hydroxy, nitrile, carboxyl, aminosulfonyl and alkylsulfonyl;
R5 is selected from the group consisting of hydrogen, lower alkyl, hydroxy and diloweralkylamino;
Y is selected from the group consisting of CHxe2x80x94OH and Cxe2x95x90O;
m is an integer from 0-2;
X is selected from the group consisting of CH2, Cxe2x95x90O and SO2; and
n is either 0 or 1
More preferred compounds of this invention include those wherein
R1 and R2 are both lower alkyl;
R3 is selected from the group consisting of phenyl, pyridinyl, pyrazinyl, pyrimidinyl, and triazinyl, wherein said substituents are one to three independently selected from the group consisting of lower alkyl, lower alkoxy, di-lower-alkylamino, aminosulfonyl and alkyl sulfonyl;
R4 is selected from the group consisting of substituted or unsubstituted phenyl, pyridinyl, pyrazinyl, pyrimidinyl, morpholinyl, triazinyl, thiophenyl, and lower alkyl, wherein said substituents are one to three independently selected from the group consisting of lower alkoxy, di-lower-alkylamino, aminosulfonyl and alkylsulfonyl.
R5 is selected from the group consisting of hydrogen and lower alkyl;
Y is Cxe2x95x90O;
M is 0 or 1;
X is CH2; and
n=0
The present invention is also a method of treating individuals with neoplastic lesions by administering a pharmacologically effective amount of an enterically coated pharmaceutical composition that includes compounds of this invention.
Preferably, such compounds are administered without therapeutic amounts of an NSAID.
Also, the present invention is a method of inhibiting the growth of neoplastic cells by exposing the cells to an effective amount of compounds of Formula I
As used herein, the term xe2x80x9cprecancerous lesionxe2x80x9d includes syndromes represented by abnormal neoplastic, including dysplastic, changes of tissue. Examples include dysplasic growths in colonic, breast, bladder or lung tissues, or conditions such as dysplastic nevus syndrome, a precursor to malignant melanoma of the skin. Examples also include, in addition to dysplastic nevus syndromes, polyposis syndromes, colonic polyps, precancerous lesions of the cervix (i.e., cervical dysplasia), esophagus, prostatic dysplasia, bronchial dysplasia, breast, bladder and/or skin and related conditions (e.g., actinic keratosis), whether the lesions are clinically identifiable or not.
As used herein, the term xe2x80x9ccancerousxe2x80x9d refers to lesions that are malignant. Examples include malignant melanomas, breast cancer, prostate cancer and colon cancer.
As used herein, the term xe2x80x9cneoplasmxe2x80x9d refers to both precancerous and cancerous lesions and hyperplasia.
As used herein, the term xe2x80x9chaloxe2x80x9d or xe2x80x9chalogenxe2x80x9d refers to chloro, bromo, fluoro and iodo groups, and the term xe2x80x9calkylxe2x80x9d refers to straight, branched or cyclic alkyl groups and to substituted aryl alkyl groups. The term xe2x80x9clower alkylxe2x80x9d refers to C1 to C8 alkyl groups.
The term xe2x80x9clower alkoxyxe2x80x9d refers to alkoxy groups having from 1 to 8 carbons, including straight, branched or cyclic arrangements.
The term xe2x80x9clower alkylmercaptoxe2x80x9d refers to a sulfide group that is substituted with a lower alkyl group; and the term xe2x80x9clower alkyl sulfonylxe2x80x9d refers to a sulfone group that is substituted with a lower alkyl group.
The term xe2x80x9cpharmaceutically acceptable saltxe2x80x9d refers to non-toxic acid addition salts and alkaline earth metal salts of the compounds of Formula I. The salts can be prepared in situ during the final isolation and purification of such compounds, or separately by reacting the free base or acid functions with a suitable organic acid or base, for example. Representative acid addition salts include the hydrochloride, hydrobromide, sulfate, bisulfate, acetate, valerate, oleate, palmetate, stearate, laurate, borate, benzoate, lactate, phosphate, tosylate, mesylate, citrate, maleate, fumarate, succinate, tartrate, glucoheptonate, lactobionate, lauryl sulfate salts and the like. Representative alkali and alkaline earth metal salts include the sodium, calcium, potassium and magnesium salts.
It will be appreciated that certain compounds of Formula I can possess an asymmetric carbon atom and are thus capable of existing as enantiomers. Unless otherwise specified, this invention includes such enantiomers, including any racemates. The separate enantiomers may be synthesized from chiral starting materials, or the racemates can be resolved by conventional procedures that are well known in the art of chemistry such as chiral chromatography, fractional cyrstallization of diastereomeric salts and the like.
Compounds of this invention may be formulated into pharmaceutical compositions together with pharmaceutically acceptable carriers for oral administration in solid or liquid form, or for intraveneous, rectal or topical administration, although carriers for oral administration are most preferred.
Pharmaceutically acceptable carriers for oral administration include capsules, tablets, pills, powders, troches and granules. In such solid dosage forms, the carrier can comprise at least one inert diluent such as sucrose, lactose or starch. Such carriers can also comprise, as is normal practice, additional substances other than diluents, e.g., lubricating agents such as magnesium stearate. In the case of capsules, tablets, troches and pills, the carriers may also comprise buffering agents. Carriers such as tablets, pills and granules can be prepared with enteric coatings on the surfaces of the tablets, pills or granules. Alternatively, the enterically coated compound can be pressed into a tablet, pill, or granule, and the tablet, pill or granules for administration to the patient. Preferred enteric coatings include those that dissolve or disintegrate at colonic pH such as shellac or Eudraget S.
Pharmaceutically acceptable carriers include liquid dosage forms for oral administration, e.g., pharmaceutically acceptable emulsions, solutions, suspensions, syrups and elixirs containing inert diluents commonly used in the art, such as water. Besides such inert diluents, compositions can also include adjuvants such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring and perfuming agents.
Pharmaceutically acceptable carriers for topical administration include DMSO, alcohol or propylene glycol and the like that can be employed with patches or other liquid-retaining material to hold the medicament in place on the skin so that the medicament will not dry out.
Pharmaceutically acceptable carriers for rectal administration are preferably suppositories that may contain, in addition to the compounds of this invention excipients such as cocoa butter or a suppository wax, or gel.
The pharmaceutically acceptable carrier and compounds of this invention are formulated into unit dosage forms for administration to a patient. The dosage levels of active ingredient (i.e., compounds of this invention) in the unit dosage may be varied so as to obtain an amount of active ingredient effective to achieve lesion-eliminating activity in accordance with the desired method of administration (i.e., oral or rectal). The selected dosage level therefore depends upon the nature of the active compound administered, the route of administration, the desired duration of treatment, and other factors. If desired, the unit dosage may be such that the daily requirement of the active compound is in one dose, or divided among multiple doses for administration, e.g., two to four times per day.
The pharmaceutical compositions of this invention are preferably packaged in a container (e.g., a box or bottle, or both) with suitable printed material (e.g., a package insert) containing indications, directions for use, etc. The general scheme for producing compounds useful in this invention is illustrated and explained below. 
Scheme I describes the general procedure which leads to compounds of Formula I. An appropriate chloromethyl lower alkylketone (R5xe2x80x94C(O)xe2x80x94CH2xe2x80x94Cl) is added to a mixture of an appropriate di-lower alkylacetone dicarboxylate, preferably the diethylester (EtO2Cxe2x80x94CH2xe2x80x94C(O)xe2x80x94C(R1, R2)xe2x80x94CO2Et) and a lower alkylamine (R4xe2x80x94NH2), preferably in an aqueous medium. The reaction temperature is preferably maintained just below 60xc2x0 C., and after a few hours, the mixture is treated with ice-hydrochloric acid (reaction 1). The thus obtained ring-closed pyrrole (a) is acylated with an acyl halide under Friedel-Crafts reaction conditions to give the pyrrole-ester (c) (reaction 3). The 5-acyl product (c) is subjected to a hydrolysis with moderately concentrated alkali (e.g., NaOH) to yield the free di-acid (d) (reaction 4), which is partly reesterified with an acidic solution of ethanol to give the 5-acyl-3-carboxy-pyrrole-ester derivative (e) (reaction 5). Decarboxylation of the carboxy group in 3-position is accomplished by heating (e) in a suitable organic solvent (reaction 6) to yield the alkyl 5-acyl-pyrrole-ester-derivative (f), which is hydrolysed (e.g., with NaOH) to give the free acid (i) (reaction 10). The pyrrole acid derivative (i) is subjected to N-bromosuccunimide to yield the lactone (j) (reaction 11).
In an alternative way to achieve the lactone (j), the acyl rest in 5-position is added at a later stage. Starting with the pyrrole derivative (a), hydrolysis in the usual manner (e.g., with NaOH; see, reaction 2) gives the dicarboxylic acid (b). Partial reesterification with an acidic solution of ethanol yields the 3-carboxy-pyrrole-ester derivative (g) (reaction 7), which is decarboxylated in the 3-position by heating it in a suitable solvent (e.g., quinoline) (reaction 9). The resulting pyrrole-ester (h) is subjected to a Friedel Crafts reaction with an acylhalide to yield the 5-acyl-pyrrole-ester derivative (f), which is converted to the lactone (j), as described above (see reactions 10 and 11). 
Scheme II is employed when R4 is hydrogen. A substituted oxime and a substituted ethylacetonedicarboxylate are allowed to react according to a Knorr pyrrole synthesis in glacial acetic acid and zinc dust to yield the ring closed pyrrole (k) (reaction 12). Hydrolysis of the diester (k) with a moderately concentrated alkali (e.g., NaOH) gives the free dicarboxylic acid (1) (reaction 13), which is re-esterified with an acidic ethanol solution (reaction 14) to yield the ethyl 5-acyl-3-carboxy pyrrole ester derivative (m). Decarboxylation of the carboxy group in 3-position is accomplished by heating the ester (m) in an organic solvent (e.g., quinoline) (reaction 15) to give the pyrrole-ester (n), which is hydrolysed in the usual manner (e.g., with NaOH) to yield the free acid (o) (reaction 16). After protecting the secondary amine by reaction with t-butyloxycarbonyl-anhydride (reaction 17), the lactone (q) is formed by reaction with N-bromosuccinimide (reaction 18). The BOC-group is removed with trifluoroacetic acid, followed by a mild basic workup (e.g., NaHCO3) to yield the lactone (r) (reaction 19).
Scheme III is employed if R4 is a group which is sensitive towards Friedel Crafts conditions, (i.e., Reactions 9 or 3 in Scheme I). The lactone (r) in a basic solution is allowed to react with an alkylhalide(R4xe2x80x94(X)n-Hal), acylhalide or sulfonylhalide to give the N-substituted lactone (s) (reaction 20), which can be reduced with sodium borohydride to the lactone (t) with a secondary alcohol in position 5 (reaction 21).
Scheme IV is employed if a nitrile, i.e., 1-methyl-pyrrole-2-acetonitrile, is available as a starting material for reaction 22, which is a Friedel Crafts acylation with R3xe2x80x94C(O)xe2x80x94Cl and AlCl3 as reagents. The nitrile (u) is hydrolysed with base (reaction 23) to give the acid (v). The lactone (s) is formed by reaction with N-bromosuccinimide (reaction 24).
To summarize, the reagents and conditions for Scheme I-III are as follows (numbers refer to reactions):
1. aqueous solution,  less than 60xc2x0 C.; H+
2. A) NaOH (25-50%)
B) HCl
3. Friedel-Crafts reaction with R3xe2x80x94C(O)xe2x80x94Cl
4. A) NaOH (25-50%)
B) HCl
5. EtOH, H+
6. xcex94, quinoline, xe2x80x94CO2↑
7. EtOH, H+ (Other lower alkylalcohols can be employed instead of ethanol.)
8. xcex94, quinoline, xe2x80x94CO2↑
9. Friedel-Crafts reaction with R3xe2x80x94C(O)xe2x80x94Cl
10. A) NaOH (25-50%)
B) HCl
11. N-bromosuccinimide
12. Knorr pyrrole synthesis conditions: Zn/CH3COOH
13. A) NaOH (25-50%)
B) HCl
14. EtOH, H+ (Other lower alkylalcohols can be employed instead of ethanol)
15. xcex94, quinoline, xe2x80x94CO2↑
16. A) NaOH (25-50%);
B) HCl
17. (BOC)2O
18. N-bromosuccinimide
19. A) CF3COOH/CH2Cl2 
B) base
20. R4xe2x80x94(X)nxe2x80x94Hal
21. NaBH4 
22. Friedel-Crafts reaction with R3xe2x80x94C(O)xe2x80x94Cl
23. 1N NaOH reflux
24. N-Bromosuccinimide